High-Moisture Meat Analogues Produced from Yellow Pea and Faba Bean Protein Isolates/Concentrate: Effect of Raw Material Composition and Extrusion Parameters on Texture Properties

Yellow pea and faba bean are potential candidates to replace soybean-based ingredients due to their suitability for cultivation in the northern hemisphere, non-genetically modified organisms cultivation practice and low risk of allergenicity. This study examined the functionality of local yellow pea and faba bean protein isolates/concentrate as meat analogue products. The most critical factors affecting the texture properties of meat analogue were also determined. Extrusion was used to produce high-moisture meat analogues (HMMAs) from yellow pea and faba bean protein isolates/concentrates and HMMAs with fibrous layered structures was successfully produced from both imported commercial and local sources. The texture properties of the HMMA produced were mainly affected by the ash, fiber and protein content and water-holding capacity of the source protein. Three extrusion process parameters (target moisture content, extrusion temperature, screw speed), also significantly affected HMMA texture. In conclusion, functional HMMA can be produced using protein isolates derived from locally grown pulses.


Introduction
High consumption of resource-intensive foods, such as animal-based products, is associated with high greenhouse gas emissions, placing a heavy burden on the food system [1]. Transition to a more sustainable diet with higher intake of plant-based foods has been identified as a key factor in improving health and reducing environmental pressure on the current food system [2,3]. Pulses are considered one of the most promising plant-based raw materials in this transition. However, consumption of locally grown pulses is low in Sweden, probably due to lack of attractive products and to anti-nutritional factors in pulses interfering with nutrient absorption [4]. There is a fast-growing market for extruded products, such as meat analogues, and extrusion technology is expected to provide various pulse-based food products that meet consumer acceptability and increase consumption of pulses in Western society.
Despite high demand for plant-based meat analogues among consumers, the texture properties remain a challenge [5,6]. Meat analogues can be produced using low-and highmoisture extrusion cooking. Products prepared by the low-moisture extrusion technique have a porous structure and the texture does not resemble animal flesh [7]. In contrast, high-moisture extrusion (HME) can create a product with a fibrous meat-like structure, Figure 1. (a) Schematic diagram of a twin-screw extruder and cooling die, modified illustration adopted from Maung & Ryu [7] and (b) cutting directions on high-moisture meat analogue (HMMA) in cutting strength measurements.

Statistical Analysis
All data were expressed as mean ± standard deviation (SD) (n = 3). One-way ANOVA and post-hoc Tukey's test were used to determine significant differences in proximate composition and WHC between the different protein isolates/concentrate. The level of significance was set to 0.05. ANOVA analyses were performed using Graphpad Prism software version 7.04-2017. Multivariate analysis, principal component analysis (PCA) and partial least square analysis (PLS) were performed using Simca software version 16.0.1. PCA was used to identify similarities or groupings in the data and to determine how the protein isolates/concentrate composition and extrusion parameters correlated to the texture properties of the HMMA. PLS was used as a supervised technique to identify the correlation between protein isolate/concentrate composition, extrusion parameters (as input X) and HMMA texture properties (as output Y). The orthogonal partial least squares (OPLS) model was used to determine the correlation between input X to one specific texture parameter (Y). The most important factors affecting HMMA texture were determined from the variable of importance (VIP ≥ 1) plot.

Characterisation of Protein Isolates/Concentrate from Pulses
There were significant differences (p < 0.05) in proximate composition between the protein isolates/concentrate. Commercial faba bean concentrate (FBC-com) had the lowest protein content of all samples (56% wb), while local faba bean isolate (FBI-local) had the highest (88% wb) ( Table 1). This was because FBC-com was produced using a dry fractionation technique that gave lower protein purity than the wet protein isolation technique [31] used for the local pulses and the commercial yellow pea protein isolate (YPIcom). Moreover, FBC-com had the highest carbohydrate and fiber content also due to the fractionation method used. Although FBC-com was prepared from dehulled grains, the fiber content was very high as compared with the other samples (Table 1). Insoluble fiber should have been removed during dehulling. However, the soluble fiber fraction might not completely detach from the protein, leading to a high fiber content in the dry-fractionated protein [32]. In contrast, the starch and fiber fractions were separated in two stages during the wet isolation technique [31]. The fat content of YPI-com and FBI-local was negligible, whereas YPI-local and FBC-com had a fat content around 3% wb ( Table 1). The high fat content in FBC-com might be due to incomplete separation of fat from the protein Due to the limited availability of protein isolates from local pulses, preliminary trials using commercial yellow pea protein isolate (YPI-com) were conducted to define the experimental settings for the extrusion parameters. In the first preliminary trial, to determine the extrusion temperature range, different temperatures above the denaturation temperature of the isolate were explored with the focus on heating zones 3 and 4 (Z3 and Z4) (Appendix B, Table A1). Thereafter, different levels of target moisture content (66-70%) and screw speed (400, 600, 800 rpm) were investigated (Appendix B, Table A3), until fibrous structures were formed in the extruded material exiting the cooling die. From the pre-study, a target moisture content below 66% resulted in jammed and burnt materials in the extruder, whereas a target moisture content above 70% did not result in a compact product. Selected extrusion parameters were also varied for the commercial faba bean protein concentrate (FBC-com) (Appendix B, Table A3). The HMMA samples from each trial stage were subjected to texture analysis (below) and compared with the reference products (boiled chicken breast, boiled beef, and commercial HMMA from soybean). The best combination of target moisture content, screw speed and extrusion temperature that resulted in a fibrous structure was then used to produce HMMA from the protein isolates from local yellow pea and faba bean (YPI-local, FBI-local) (Appendix B, Table A3). Specific mechanical energy (SME, kJ/kg) was calculated using the formula [30]: where n is the screw speed (400, 600, 800 rpm), T is torque (obtained from % drive load × 80 Nm) and MFR is mass flow rate (2 kg/h).

Texture Properties of HMMA
Texture profile analysis (TPA) of the HMMA obtained was carried out in triplicate, using a texture analyzer (TVT-300XP, Perten Instruments AB, Hägersten, Sweden), according to Zahari et al. (2020) [28]. The meat analogue samples were cut and shaped into 20 mm × 20 mm pieces (7 mm thickness), and then compressed by 2 mm from initial height using a cylindrical probe (diameter 18 mm). The hardness, springiness and chewiness of the samples were recorded. A second analysis was carried out with a cutting test used previously to assess fibrous structure formation in meat analogues [8,16]. The cutting strength was evaluated using a knife blade (height 117 mm) penetrating the sample (20 mm × 20 mm, 7 mm thickness) to 5 mm depth at a speed of 2 mm/s. Triplicate samples were cut longitudinally (lengthwise) and transverse (crosswise) to the outflow direction from the extruder, as depicted in Figure 1b. Similar values in longitudinal and transverse cuts indicate that the meat analogue sample has a uniform texture, with no fibrous layered structure [8].

Statistical Analysis
All data were expressed as mean ± standard deviation (SD) (n = 3). One-way ANOVA and post-hoc Tukey's test were used to determine significant differences in proximate composition and WHC between the different protein isolates/concentrate. The level of significance was set to 0.05. ANOVA analyses were performed using Graphpad Prism software version 7.04-2017. Multivariate analysis, principal component analysis (PCA) and partial least square analysis (PLS) were performed using Simca software version 16.0.1. PCA was used to identify similarities or groupings in the data and to determine how the protein isolates/concentrate composition and extrusion parameters correlated to the texture properties of the HMMA. PLS was used as a supervised technique to identify the correlation between protein isolate/concentrate composition, extrusion parameters (as input X) and HMMA texture properties (as output Y). The orthogonal partial least squares (OPLS) model was used to determine the correlation between input X to one specific texture parameter (Y). The most important factors affecting HMMA texture were determined from the variable of importance (VIP ≥ 1) plot.

Characterisation of Protein Isolates/Concentrate from Pulses
There were significant differences (p < 0.05) in proximate composition between the protein isolates/concentrate. Commercial faba bean concentrate (FBC-com) had the lowest protein content of all samples (56% wb), while local faba bean isolate (FBI-local) had the highest (88% wb) ( Table 1). This was because FBC-com was produced using a dry fractionation technique that gave lower protein purity than the wet protein isolation technique [31] used for the local pulses and the commercial yellow pea protein isolate (YPI-com). Moreover, FBC-com had the highest carbohydrate and fiber content also due to the fractionation method used. Although FBC-com was prepared from dehulled grains, the fiber content was very high as compared with the other samples (Table 1). Insoluble fiber should have been removed during dehulling. However, the soluble fiber fraction might not completely detach from the protein, leading to a high fiber content in the dryfractionated protein [32]. In contrast, the starch and fiber fractions were separated in two stages during the wet isolation technique [31]. The fat content of YPI-com and FBI-local was negligible, whereas YPI-local and FBC-com had a fat content around 3% wb ( Table 1). The high fat content in FBC-com might be due to incomplete separation of fat from the protein fractions during dry-fractionation [33]. As YPI-local and FBI-local were prepared using the same method, the difference in lipid retention during the extraction might be due to different lipid composition and location in the grain matrix [34]. Different numbers of endothermic peaks were observed for yellow pea and faba bean proteins (Appendix B, Figure A2), four peaks identified in YPI-com (67 • C, 88 • C, 102 • C, 132 • C) and three in YPI-local (72 • C, 89 • C, 116 • C) (Appendix B, Figure A2). Furthermore, FBC-com had three endothermic peaks (77 • C, 95 • C, 128 • C), whereas FBI-local only had one peak at 97 • C (Appendix B, Figure A2). The first endothermic peak for both yellow pea and faba bean proteins is assumed to be the starch gelatinization peak, as observed by doCarmo et al. [32]. The main endothermic peak at 88-89 • C for yellow pea and at 95-97 • C for faba bean might correspond to the denaturation temperature of globulins which are the major storage proteins in pulses [32,35]. The highest melting temperature was 132 • C for yellow pea protein and 128 • C for faba bean protein. This indicates that the cooking temperature zone in the extruder should be set above 132 • C to achieve a complete melt of the food mix which is critical for the production of meat analogue products [8].
The YPI-com, YPI-local and FBI-local had similar WHC (3 mL/g), which were around three-fold higher than that of FBC-com (1 mL/g) ( Table 1). According to Lam et al. [36], proteins are more denatured during the wet isolation process, leading to changes in tertiary and quaternary protein structure. These structural changes may increase exposure of hydrophilic groups, in turn increasing the ability to hold water. WHC is an important functional property in food formulation, due to the effect on the texture of the final product [37].
In terms of color, FBI-local was dark grey, whereas the commercial protein concentrate was light-colored ( Figure 2). According to information from the supplier, FBC-com was made using dehulled faba bean grains as the raw material, whereas for FBI-local the whole faba bean grains, with the hull intact, were used. It was observed in the leaching phase that the color of the dispersion gradually became darker over time during the incubation for 1 h. Based on Sharan et al. [35], this could be due to the content of proanthocyanidins in faba bean hull being subjected to oxidation. The color of the FBI-local protein precipitate after pH adjustment with acid was light grey, but it changed to dark grey when the precipitate was neutralized (pH 7) just before freeze-drying. Thus, the precipitate could be freeze-dried directly after acidification to avoid dark coloration of the protein isolate, although there is the risk of an acidic flavor if the pH is not neutralized.

Preliminary Extrusion Trials
In preliminary trials using YPI-com to define the extrusion parameter settings (extrusion temperature, screw speed, target moisture content), the temperature in the four heating zones (Z1-Z4) in the extruder barrel was adjusted individually. The temperature in Z1 and Z2 was kept at 40 and 80 °C, respectively, while in Z3 and Z4 four combinations of extrusion temperatures ranging from 110-150 °C were tested (Appendix B, Table A1).
The texture of the extrudate was affected by the temperature in Z3 and Z4, with

Preliminary Extrusion Trials
In preliminary trials using YPI-com to define the extrusion parameter settings (extrusion temperature, screw speed, target moisture content), the temperature in the four heating zones (Z1-Z4) in the extruder barrel was adjusted individually. The temperature in Z1 and Z2 was kept at 40 and 80 • C, respectively, while in Z3 and Z4 four combinations of extrusion temperatures ranging from 110-150 • C were tested (Appendix B, Table A1).
The texture of the extrudate was affected by the temperature in Z3 and Z4, with higher temperature giving extrudates with reduced hardness and chewiness. However, there was no apparent effect of extrusion temperatures on the cutting strength of HMMA (Appendix B, Table A2). At an extrusion temperature of 110-110 • C in Z3-Z4, the samples were compact, but no fibrous structures were formed. With increased extrusion temperature in Z3-Z4, fibrous layered structures started to form and were most evident at 130-150 • C on visual inspection. Others have observed a similar effect of extrusion temperature on fiber formation, in extrudates produced from pea and soy protein, respectively [8,38]. It has been suggested that, in order to obtain layered fiber in extrudates, the food mix (e.g., protein, carbohydrate, water) has to be completely melted in the extruder barrel [8,38]. At too low extrusion temperature, the food mix is only partly melted and non-melted molecules may disperse in the matrix, causing poor layered fibrous formation. An increased extrusion temperature will induce complete melting of the food mix, with reduced viscosity and initiation of layered fibrous structure formation. The texture of the extrudates was also affected by the target moisture content, with a higher moisture content giving a softer extrudate (Appendix B, Table A2).
A decrease in specific mechanical energy (SME) was observed as the extrusion temperature increased (Appendix B, Table A2), confirming findings by others [30,39]. This was due to lower viscosity of the material in the barrel as a result of higher extrusion temperature [30]. SME reflects the mechanical work put into the material during extrusion [33] and can affect final product characteristics such as hardness, density and expansion index [16].
Differential scanning calorimetry (DSC) analysis of YPI-com indicated that its highest melting temperature was 132 • C (Appendix B, Figure A2). To achieve complete melting of the food mix and facilitate fibrous structure formation in the HMMA, the extruder should therefore be operated above 132 • C when processing yellow pea protein isolate. The chosen extrusion temperature profile for YPI-com was 40-80-130-150 • C (Z1-Z2-Z3-Z4). This temperature profile was also used to produce HMMA from FBC-com, but a minor adjustment was needed in Z2 to prevent backflow during extrusion. Thus, the chosen extrusion temperature profile for FBC-com (Z1-Z2-Z3-Z4) was 40-60-130-150 • C.

HMMA Production Using Local Pulse Protein Isolates
In terms of protein content, YPI-local contained 81% protein, which was similar to the level in YPI-com (79%) ( Table 1). Thus, production of HMMA from YPI-local followed the extrusion parameters for YPC-com. HMMA samples with prominent layered fibrous structure ( Figure 3) were successfully produced from YPI-local at: 67% target moisture content, extrusion temperature 40-80-130-150 • C (Z1-Z2-Z3-Z4) and screw speed 400 and 600 rpm (Appendix C, Table A4). No sample was obtained at screw speed 800 rpm, due to unstable flow of output.

Effect of Protein Isolate/Concentrate Composition and Extrusion Parameters on HMMA Texture Properties
Unsupervised PCA revealed different groupings of HMMA samples based on the raw material (coefficient of determination R2X(cum) = 0.75, coefficient of prediction Q2(cum) = 0.65) (figure not shown). The PLS model, using protein isolate/concentrate composition and extrusion parameters as X-variables and texture properties of HMMA as Y-variables, resulted in a coefficient of determination (R2Y(cum)) of 0.50 and the coefficient of prediction (Q2(cum)) of 0.38 (Figure 4a).
In the PLS bi-plot (Figure 4a), the HMMA samples made from FBC-com and FBI-local were located in separate quadrants, indicating a difference in texture properties compared with HMMA samples made from YPI-com and YPI-local, which were more similar in terms of texture. As indicated by the variable of importance plot (VIP) for the PLS model, the variables that were most important for the separation of the different HMMA samples in the PLS model were target moisture content and temperature in Z2 (Figure 4b). Ash, fiber, fat and protein content and WHC of the protein isolates/concentrate were also important for the texture of the HMMA. For the specific texture parameter hardness, the OPLS model suggested that the hardness of HMMA was positively correlated with ash and fiber content of the protein isolate/concentrate, but negatively correlated with WHC and target moisture content of the protein isolate/concentrate, when adding all four raw materials to the same model (Figure 4c). For FBI-local, the extrusion parameters used for YPI-com and FBC-com were initially applied. However, these settings led to backflow and jammed material in the cooling die, so the temperature in Z3 and Z4 had to be decreased to allow consistent flow. HMMA with layered fibrous structure (Figure 3) was successfully produced from FBI-local at: 62% target moisture content, extrusion temperature 40-60-110-130 • C (Z1-Z2-Z3-Z4) and screw speed 800 rpm (Appendix C, Table A4). Proteins with low denaturation temperature do not require high extrusion temperature and can easily be denatured and texturized by pressure and shear in the extruder [17]. FBI-local had a protein denaturation peak at 97 • C (Appendix B, Figure A2) which might explain why a fibrous structure formed even though the extrusion temperature was lower. Thus, the runs confirmed that HMMA could be produced from yellow pea and faba bean proteins (both commercial and local). Some modifications were needed in the temperature zones, screw speed and target moisture content to create a layered fibrous structure and ensure a consistent flow of extrudates.
The HMMA produced from yellow pea and faba bean was significantly (p < 0.05) harder compared to the reference commercial soybean HMMA, but similar to the boiled chicken and beef depending on extruder settings (Appendix C, Table A4). The springiness did not differ (p > 0.05) between the HMMA produced and the references, independent on origin. The commercial soybean HMMA had significantly (p < 0.05) lower chewiness than any of the HMMA produced as well as the chicken and beef reference. The HMMA produced from yellow pea had similar chewiness as the boiled chicken and beef, depending on the extruder setting. In contrast, the HMMA produced from faba bean, independent of the extruder settings, was significantly chewier than the references.
HMMA produced from YPI-com, YPI-local and FBC-com had higher crosswise than lengthwise cutting strength (Appendix C, Table A4). This indicates that the fibers were more textured in the longitudinal direction. In contrast, HMMA made from FBI-local had higher lengthwise than crosswise cutting strength (Appendix C, Table A4). Hence, the fibers in FBI-local were more aligned in the crosswise direction (parabolic pattern), resembling those in the reference samples (boiled chicken, boiled beef, commercial soy HMMA). In extrusion of HMMA from pea protein with 55% moisture content at 130 • C, Osen et al. [8] also observed layered fibrous structures in parabolic patterns. Upon raising the extrusion temperature to 160 • C, they found that a predominantly lengthwise fibrous structure appeared [8], as also observed in the present study. Osen et al. [8] suggested that the melt viscosity decreases as the extrusion temperature increases, affecting the flow pattern in the cooling die. The higher temperature leads to higher flow velocity at the core of the flow profile, which upon solidification might lead to more lengthwise-oriented fibers [8].

Effect of Protein Isolate/Concentrate Composition and Extrusion Parameters on HMMA Texture Properties
Unsupervised PCA revealed different groupings of HMMA samples based on the raw material (coefficient of determination R2X(cum) = 0.75, coefficient of prediction Q2(cum) = 0.65) (figure not shown). The PLS model, using protein isolate/concentrate composition and extrusion parameters as X-variables and texture properties of HMMA as Y-variables, resulted in a coefficient of determination (R2Y(cum)) of 0.50 and the coefficient of prediction (Q2(cum)) of 0.38 (Figure 4a).  In the PLS bi-plot (Figure 4a), the HMMA samples made from FBC-com and FBI-local were located in separate quadrants, indicating a difference in texture properties compared with HMMA samples made from YPI-com and YPI-local, which were more similar in terms of texture. As indicated by the variable of importance plot (VIP) for the PLS model, the variables that were most important for the separation of the different HMMA samples in the PLS model were target moisture content and temperature in Z2 (Figure 4b). Ash, fiber, fat and protein content and WHC of the protein isolates/concentrate were also important for the texture of the HMMA. For the specific texture parameter hardness, the OPLS model suggested that the hardness of HMMA was positively correlated with ash and fiber content of the protein isolate/concentrate, but negatively correlated with WHC and target moisture content of the protein isolate/concentrate, when adding all four raw materials to the same model (Figure 4c).

Effects of Protein Isolate/Concentrate Composition
As suggested by the PLS and the OPLS models shown in Figure 4, the ash content of the protein isolates/concentrate was an important factor affecting the texture of HMMA, with higher ash content correlated with harder texture. Calcium ions have previously been found to play an essential role in forming a fibrous layered texture in a meat analogue from caseinate mixture, whereas a sodium caseinate mixture did not result in any fibrous structure [40]. Formation of fibrous structure in that study was attributed to the presence of divalent calcium ions causing stronger interactions between the caseinate micelles, resulting in aggregation and better texturization of the meat analogue [37]. Thus, it appears that disulphide bonds are not the only important factor in the formation of the fibrous structure during extrusion, and that there is also a contribution from ionic bonds. Faba bean contains higher amounts of calcium, phosphorus, iron and zinc than yellow pea [41,42], so when faba bean was extruded using the same extrusion parameters and the same target moisture content as yellow pea, the hardness was significantly higher (2578 g) than that found for yellow pea (1760 g) (Appendix C, Table A4). This difference might be due to the higher mineral content in the faba bean isolate/concentrate contributing more ionic bonds, which in turn increased the hardness of HMMA.
The hardness of HMMAs were found to be positively correlated with total dietary fiber and fat content of the protein isolates/concentrate and negatively correlated with the protein content ( Figure 4c, Table 1). FBC-com had the lowest protein content of the raw materials investigated, and the corresponding HMMA had the highest values for hardness and chewiness (Appendix C, Table A4). FBC-com was produced using a dry fractionation technique that resulted in lower protein content and higher content of fat, fiber and carbohydrate than the protein isolates investigated (Table 1). Pietsch et al. [43] concluded that when the raw material contains a substantial amount of non-nitrogenous components (such as polysaccharides), the rheological properties of the mix during extrusion and texturization of HMMA are more influenced by protein-polysaccharide interactions than by protein-protein interactions. High extrusion temperature may enhance the compatibility of proteins and polysaccharides, thus reducing the microstructural interface between them and leading to a product with higher rigidity, but lower texturization [13,44,45], as also found in the present study.
Another important factor affecting the texture of HMMA, according to the OPLS model, was the WHC of the protein isolates/concentrate, with lower WHC correlated to higher hardness (Figure 4c). WHC depends on the conformation of proteins, the number of hydrophilic sites and carbohydrate content [46]. Lower WHC might indicate that more proteins are in their native state [36]. However, upon hydration, heating and shearing in the extruder, the native proteins start to unfold, resulting in increased interaction between the proteins and other components, such as polysaccharides. In the present study, FBC-com had significantly lower WHC than the other three isolates investigated. HMMA produced from FBC-com also had a firmer texture in general, which can be partly explained by its low capacity to hold water.

Effect of Extrusion Parameters
The texture properties and cutting strength of HMMAs decreased as the target moisture content increased (Appendix C, Table A4). This is agreement with findings by others of a decrease in hardness, chewiness and cutting strength in HMMA made from soy and lupin protein with increasing moisture content [18,30]. At higher moisture content, the viscosity and temperature of the mix in the barrel decreases, causing incomplete protein denaturation and therefore less protein interaction and cross-linking, leading to a softer product [18,30]. YPI-com and FBC-com required a different target moisture content to produce the meat analogue (Appendix C, Table A4). HMMA samples produced from FBC-com with moisture content above 62% were very compact, with a fudge-like texture and no layered fibers, possibly due to the much lower protein content (55% wb) in FBC-com compared with YPI-com (79% wb) ( Table 1). Protein interactions and cross-linking are prerequisites to create the layered fibrous structure of HMMA, and hence a lower moisture content was needed to produce HMMA from FBC-com.
The extrusion temperature in Z2 appeared to be an important variable affecting the texture of HMMA (Figure 4b). The same extrusion temperature in Z2 (60 • C) was used to produce HMMA from FBI-local and FBC-com. HMMA samples produced from both commercial and local faba bean proteins had higher values in all texture properties compared with HMMA made from yellow pea proteins (Appendix C, Table A4). This might be due to differences in protein composition and properties (Appendix B, Figure A2) between faba bean and yellow pea [47].
The effect of different screw speeds on texture properties and cutting strength of HMMA was not consistent (Appendix C, Table A4). It was observed that screw speed affected the hardness and cutting strength to a greater extent at higher moisture contents (69-70%). In this moisture range, higher screw speed led to an increased degree of texturization, and thus higher values of hardness and cutting strength (crosswise and lengthwise). At lower moisture contents (≤68%), there was no clear relationship between screw speed and HMMA texture parameters (Appendix C, Table A4). This finding was supported by the multivariate statistical analysis, where screw speed had an impact on the texture parameters ( Figure 4b). However, the error bar was high, indicating that screw speed was an essential factor for some HMMA formulations investigated, but not for others. Zhang et al. [48] found that increased screw speed (from 250 to 350 rpm) led to an increased degree of texturization of vegetable protein from peanut, but that when the screw speed increased further (450 rpm), the strong shear force led to a weaker fibrous structure. Palanisamy et al. [30] found that a higher screw speed led to higher cutting strength of HMMA from lupin protein, due to increased cross-linking and polymerization. These differences between studies may be due to differences in the raw material and extrusion parameters used.

Conclusions
High-moisture extrusion cooking was successfully applied to produce HMMAs with fibrous layered structures from yellow pea and faba bean proteins, both imported commercial and local. The extruder processing parameter settings had to be adjusted for the different types of protein isolate/concentrate, e.g., a lower target moisture content was needed for faba bean proteins compared with yellow pea proteins. The most important factors affecting the texture properties of HMMAs in this study were the ash, fiber, fat and protein content and water-holding capacity of the protein isolate/concentrate. Studied extrusion parameters affected the texture of HMMA. Further studies on the effect of different screw configurations on the fibrous structure formation could be explored, as well as sensory studies regarding the textural properties and acceptability of the HMMAs.   Figure A2. Denaturation temperature and enthalpy of (a) yellow pea isolate and (b) faba bean isolate. Figure A2. Denaturation temperature and enthalpy of (a) yellow pea isolate and (b) faba bean isolate.